Experiment one Examination of Bacteria

Visualizing Bacteria Staining is required to properly visualize bacteria

Microscopy Bright Field Microscopy

Microscopy Bright Field Microscopy Three different objective lenses are commonly used 10x: To scan the slide for specimens 40x: To view parasites, filamentous fungi 100x: To observe single cells Total magnification= (objective lens magnification) X (ocular lens magnification)

Microscopy Bright Field Microscopy Note how light scatters via refraction Refractive index of air is lower than that of glass Loss of refracted light is minimized by applying mineral oil (same RI as glass)

Microscopy Dark Field Microscopy Creates contrast between the object and the surrounding field. Background is dark and the object is bright. An annular stop ring permits light coming from the outside of the beam. When light from the stop is deflected and deviated by the object can it be seen. Advantageous for viewing thin bacteria (Ie. Treponema pallidum) Disadvantage: internal structure is not as clearly visable compared to bright field

Microscopy Dark Field Microscopy Light Field vs Dark Field

Microscopy Phase-Contrast Microscopy Most of the detail of living cells is undetectable in bright field microscopy Little contrast exists between structures with similar transparency Insufficient natural pigmentation. Organelles show wide variation in refractive index (the tendency of the materials to bend light) providing an opportunity to distinguish them with phase contrast mircoscopy Internal features are more easily viewed

Microscopy Phase-Contrast Microscopy

Microscopy Fluorescent Microscopy Organisms are stained with fluorescent dies (fluorochromes) and then viewed. Fluorescent microscopes emit a shorter wavelength of light than in bright field microscopy. The short wavelength excites the fluorochromes, and they fluoresce. Allows for easier low magnification scanning. Bright object occurs against a dark background.

Microscopy Fluorescent Microscopy Fluorescent vs Dark Field

Microscopy Fluorescent Microscopy Different fluorescent stains can bind to different targets Digital merging with differential stains

Electron Microscopy Uses magnetic coils to direct a beam of electrons through the specimen onto a screen Uses a very short wavelength, thus magnification and resolution is improved Samples are stained/coated with metal ions to create contrast.

Electron Microscopy Transmission electron microscope Electrons pass through the specimen

Electron Microscopy Scanning Electron Microscope Electrons bounce off the surface of a specimen and create a 3D image.

How to use the oil immersion lens of microscopy The substage condenser to raise to the highest position The iris diaphragm fully opened To adjust the light entering the lens with low-power lens The oil immersion lens to be rotated into position The specimen to be put on the center of stage A drop of oil to be placed on the slide directly over the area to be viewed

Using the fine adjustment knob, to bring the specimen into sharp focus Up the stage with the coarse adjustment knob, to let oil lens into the oil Looking into the ocular lens and down the stage slowly with the coarse adjustment knob until the specimen comes into focus Using the fine adjustment knob, to bring the specimen into sharp focus The oil immersion lens should be cleaned with lens paper after experiment

Examination Methods: Direct Examination India Ink Darkens the background rather than the cell Useful in detecting Cryptococcus capsules Capsule excludes ink

Morphological Observation Of Bacterial Cells S. aureus

Morphological Observation Of Bacterial Cells E. coli

Morphological Observation Of Bacterial Cells V. choleriae Gram -ve

Morphological Observation Of Bacterial Cells Streptococcus pneumoniae

Morphological Observation Of Bacterial Cells Salmonella typhi

Morphological Observation Of Bacterial Cells Clostridium tetani Club shape is due to endospore production at one termini of the cell.

Staining of Bacteria PURPOSE : To make bacteria more easily observable To acquaint you with Gram stain MATERIALS: Simple stain Gram stain Acid-fast stain Special stain Spore stain Capsule stain Flagella stain Metachromatic granules stain.

Gram stain purpose: MATERIALS : differentiating bacteria Slant cultures of and Escherichia coli and S.aureus (18 to 24 hours old) Crystal violet, iodine solution, 95% alcohol, safranin Microscope slides

Gram stain PROCEDURE: Smear: size of a dime to form a thin film Dry : air dry Fix: through the warm air above the flame two or three times.

Process of Gram’s Stain Crystal violet (primary staining) Washing 1min Lugol’s iodine (mordant staining) Washing 1min 95%ethyl alcohol (decolorization) Fuchsion red (counterstaining) Washing 30s Washing 30s Blot dry with bibulous papers Observation with the oil immersion lens Results :Gram –positive blue color Gram-negative red color

Gram Staining In gram-positive bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within the cell. Gram +ve bacteria have low lipid content Lipid is dissolved by alcohol The alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan Thus causing the cell wall to trap the crystal violet-iodine complex within the cell.

Gram Staining Gram-negative bacteria have higher lipid contents The alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached. The single thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the cell is decolorized.

Gram Staining: Common Errors There are several factors that could result in a gram-positive organism staining gram-negatively: The method and techniques used. Overheating during heat fixation, over decolorization with alcohol, and even too much washing with water between steps may result in gram-positive bacteria losing the crystal violet-iodine complex. The age of the culture. Cultures more than 24 hours old may lose their ability to retain the crystal violet-iodine complex. The organism itself. Some gram-positive bacteria are more able to retain the crystal violet-iodine complex than others. Therefore, one must use very precise techniques in gram staining and interpret the results with discretion.